Functional and molecular characterization of endothelium-dependent and endothelium-independent relaxant pathways in uterine artery of non-pregnant buffaloes

  • Udayraj P. Nakade
  • Abhishek Sharma
  • Priyambada Kumari
  • Shirish Bhatiya
  • Sooraj V. Nair
  • K. N Karikaran
  • Vipin Sharma
  • Soumen Choudhury
  • Satish Kumar GargEmail author
Original Article


Present study was undertaken to unravel the endothelium-dependent and endothelium-independent relaxant pathways in uterine artery of non-pregnant buffaloes. Isometric tension of arterial rings was recorded using data acquisition system based polyphysiograph. Acetylcholine (ACh) produced endothelium-dependent vasorelaxation by releasing nitric oxide (NO), and inhibition of nitric oxide synthase (NOS) by L-NAME (300 μM) significantly (P < 0.05) reduced the NO release and thereby the vasorelaxant effect of ACh. However, L-NMMA, another NOS inhibitor, and PTIO, a NO scavenger, did not have any additional inhibitory effect on NO and ACh-induced vasorelaxation. Cyclooxygenase (COX) inhibitor (indomethacin) alone did not have any inhibitory action on vasorelaxant response to ACh; however, simultaneous inhibition of COX and NOS enzymes significantly (P < 0.05) attenuated the relaxant response indicating the concurrent release of these two mediators in regulating ACh-induced relaxation. Besides NOS and COX-derived metabolites (EDRF), small (SKCa) and intermediate (IKCa) conductance K+ channels being the members of EDHF play predominant role in mediating ACh-induced vasorelaxation. Using different molecular tools, existence of eNOS, COX-1, and,IKCa in the endothelium, BKCa in vascular smooth muscle, and SKCa in both endothelium and vascular smooth muscle was demonstrated in buffalo uterine artery. Gene sequencing of COX-1 and SKCa genes in uterine artery of buffaloes showed more than 97% structural similarity with ovine (Ovis aries), caprine (Capra hircus), and Indian cow (Bos indicus). Endothelium-independent nitrovasodilator, sodium nitroprusside (SNP), produced vasorelaxation which was sensitive to blockade by soluble guanylate cyclase (sGC) inhibitor (ODQ), thus suggesting the important role of cGMP/PKG pathways in uterine vasorelaxation in buffaloes. Taken together, it is concluded that both endothelium-dependent (EDHF and EDRF) and endothelium-independent (sGC-cGMP) relaxant pathways are present in uterine arteries of non-pregnant buffaloes, and they differently contribute to vasorelaxation during non-pregnant state.


Nitric oxide Cyclooxygenase K+ channels Uterine artery Non-pregnant Buffalo 


Authors’ contributions

UPN, AS, and VS conducted functional experiments; UPN, SC, PK, SVN, and KKN conducted molecular experiments; SC and SKG designed the research; UPN, SC and SB analyzed the data, and SC and SKG wrote the manuscript.

Funding information

Research work presented in this manuscript was supported by Indian Council of Agricultural Research, New Delhi, India under Niche Area of Excellence Programme (Grant No. 10 (10)/2012-EPD dated 23 March 2012) to Department of Veterinary Pharmacology and Toxicology, DUVASU, Mathura, India. Financial assistance by ICAR is thankfully acknowledged. The first author was also awarded the INSPIRE fellowship from Department of Science & Technology (DST-INDIA) via Grant No. DST/INSPIRE Fellowship/2014/IF140997.

Compliance with ethical standards

Conduct of experiments on uterus, which are non-edible and thrown away by the slaughterers, was collected from the slaughterhouse or butcher’s shops, and these are not governed by the “Guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals” of the Government of India as resolved by the Institute Animal Ethics Committee in its meeting held on 16 September 2016 (Institutional Animal Ethics Committee; Approval No. 110/IAEC/16/40/2).

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

210_2019_1726_MOESM1_ESM.docx (39 kb)
ESM 1 (DOCX 38 kb)


  1. Ali A, Abdel‐Razek AK, Abdel‐Ghaffar S, Glatzel PS (2003) Ovarian follicular dynamics in buffalo cows (Bubalus bubalis). Reprod Domest Anim 38(3):214–218Google Scholar
  2. Beverelli F, Bea ML, Puybasset L, Giudicelli JF, Berdeaux A (1997) Chronic inhibition of NO synthase enhances the production of prostacyclin in coronary arteries through upregulation of the cyclooxygenase type 1 isoform. Fundam Clin Pharmacol 11(3):252–259CrossRefGoogle Scholar
  3. Bird IM, Zhang L, Magness RR (2003) Possible mechanisms underlying pregnancy-induced changes in uterine artery endothelial function. Am J Phys Regul Integr Comp Phys 284(2):R245–R258Google Scholar
  4. Boura ALA, Walters WAW, Read MA, Leitch IM (1994) Autacoids and control of human placental blood flow. Invited review Clin Exp Pharmacol Physiol 21:737–748Google Scholar
  5. Carbillon L, Perrot N, Uzan M, Uzan S (2001) Doppler ultrasonography and implantation: A critical review. Fetal Diagn Ther 16(6):327–332CrossRefGoogle Scholar
  6. Choudhury S, Kannan K, Pule Addison M, Darzi SA, Singh V, Singh TU, Thangamalai R, Dash JR, Parida S, Debroy B, Paul A, Mishra SK (2015) Combined treatment with atorvastatin and imipenem improves survival and vascular functions in mouse model of sepsis. Vasc Pharmacol 71:139–150CrossRefGoogle Scholar
  7. Cohen RA, Plane F, Najibi S, Huk I, Malinski T, Garland CJ (1997) Nitric oxide is the mediator of both endothelium-dependent relaxation and hyperpolarization of the rabbit carotid artery. Proc Natl Acad Sci U S A 94(8):4193–4198CrossRefGoogle Scholar
  8. Cooke CL, Davidge ST (2003) Pregnancy-induced alterations of vascular function in mouse mesenteric and uterine arteries. Biol Reprod 68(3):1072–1077CrossRefGoogle Scholar
  9. Dalle Lucca JJ, Adeagbo AS, Alsip NL (2000) Oestrous cycle and pregnancy alter the reactivity of the rat uterine vasculature. Hum Reprod 15(12):2496–2503CrossRefGoogle Scholar
  10. Eckman DM, Gupta R, Rosenfeld CR, Morgan TM, Charles SM, Mertz H, Moore LG (2012) Pregnancy increases myometrial artery myogenic tone via NOS- or COX-independent mechanisms. Am J Phys Regul Integr Comp Phys 303(4):R368–R375Google Scholar
  11. Fouty B, Komalavilas P, Muramatsu M, Cohen A, McMurtry IF, Lincoln TM, Rodman DM (1998) Protein kinase G is not essential to NO-cGMP modulation of basal tone in rat pulmonary circulation. Am J Phys 274(2 Pt 2):H672–H678Google Scholar
  12. Friedler S, Schenker JG, Herman A, Lewin A (1996) The role of ultrasonography in the evaluation of endometrial receptivity following assisted reproductive treatments: a critical review. Hum Reprod Update 2:323–335CrossRefGoogle Scholar
  13. Gambone LM, Murray PA, Flavahan NA (1997) Synergistic interaction between endothelium-derived NO and prostacyclin in pulmonary artery: potential role for K+ATP channels. Br J Pharmacol 121:271–279CrossRefGoogle Scholar
  14. Gangula PR, Zhao H, Supowit S, Wimalawansa S, DiPette D, Yallampalli C (1999) Pregnancy and steroid hormones enhance the vasodilation responses to CGRP in rats. Am J Phys 276(1 pt 2):H284–H288Google Scholar
  15. Gauthier KM, Spitzbarth N, Edwards EM, Campbell WB (2004) Apamin-sensitive K+ currents mediate arachidonic acid-induced relaxations of rabbit aorta. Hypertension. 43:413–419CrossRefGoogle Scholar
  16. Gebremedhin D, Kaldunski M, Jacobs ER, Harder DR, Roman RJ (1996) Co-existence of two types of calcium activated potassium channels in rat renal arterioles. Am J Phys 270:F69–F81Google Scholar
  17. Gillham JC, Myers JE, Baker PN, Taggart MJ (2007) Regulation of endothelial-dependent relaxation in human systemic arteries by SKCa and IKCa channels. Reprod Sci 14:43–50CrossRefGoogle Scholar
  18. Gokina NI, Kuzina OY, Vance AM (2010) Augmented EDHF signaling in rat uteroplacental vasculature during late pregnancy. Am J Physiol Heart Circ Physiol 299:H1642–H1652CrossRefGoogle Scholar
  19. Golding EM, Ferens DM, Marrelli SP (2002) Altered calcium dynamics do not account for attenuation of endothelium-derived hyperpolarizing factor-mediated dilations in the female middle cerebral artery. Stroke. 33:2972–2977CrossRefGoogle Scholar
  20. Habermehl DA, Janowiak MA, Vagnoni KE, Bird IM, Magness RR (2000) Endothelial vasodilator production by uterine and systemic arteries. IV Cyclooxygenase isoform expression during the ovarian cycle and pregnancy in sheep. Biol Reprod 62:781–788CrossRefGoogle Scholar
  21. Hu XQ, Xiao D, Zhu R, Huang X, Yang S, Wilson S, Zhang L (2011) Pregnancy upregulates large-conductance Ca(2+)-activated K(+) channel activity and attenuates myogenic tone in uterine arteries. Hypertension. 58:1132–1139CrossRefGoogle Scholar
  22. Itoh H, Bird IMI, Nakao K, Magness RR (1998) Pregnancy increases soluble and particulate guanylate cyclases and decreases the clearance receptor of natriuretic peptides in ovine uterine but not systemic arteries. Endocrinology. 138:3329–3341CrossRefGoogle Scholar
  23. Jovanović A, Grbović L, Jovanović S (1995) Effect of the vascular endothelium on noradrenaline-induced contractions in non-pregnant and pregnant Guinea-pig uterine arteries. Br J Pharmacol 114(4):805–815CrossRefGoogle Scholar
  24. Jovanović A, Grbović L, Drekić D, Novaković S (1994) Muscarinic receptor function in the Guinea-pig uterine artery is not altered during pregnancy. Eu J Pharmacol 258(3):185–194CrossRefGoogle Scholar
  25. Kopp L, Paradiz G, Tucci JR (1977) Urinary excretion of cyclic 3′,5′-adenosine monophosphate and cyclic 3′, 5′-guanosine monophosphate during and after pregnancy. J Clin Endocrinol Metab 44:590–594CrossRefGoogle Scholar
  26. Kovitz K, Aleskowitch TD, Sylvester JT, Flavahan NA (1993) Endothelium derived contracting and relaxing factors contribute to hypoxic responses of pulmonary arteries. Am J Phys 256:H1139–H1148Google Scholar
  27. Ledoux J, Werner ME, Brayden JE, Nelson MT (2006) Calcium-activated potassium channels and the regulation of vascular tone. Physiology (Bethesda) 21:69–78Google Scholar
  28. Leo MD, Siddegowda YK, Kumar D, Tandan SK, Sastry KV, Prakash VR, Mishra SK (2008) Role of nitric oxide and carbonmonoxide in N (omega)-nitro-L-arginine methyl ester-resistant acetylcholine-induced relaxation in chicken carotid artery. Eur J Pharmacol 596:111–117CrossRefGoogle Scholar
  29. Lidbury PS, Antunes E, De Nucci G, Vane JR (1989) Interactions of iloprost and sodium nitroprusside on vascular smooth muscle and platelet aggregation. Br J Pharmacol 98:1275–1280CrossRefGoogle Scholar
  30. Lugnier C, Komas N (1993) Modulation of vascular cyclic nucleotide phosphodiesterases by cyclic GMP: role in vasodilatation. Eur Heart J 14(suppl. I):141–148Google Scholar
  31. Lunell NO, Nylund LE, Lewander R, Sarby B (1982) Uteroplacental blood flow in pre-eclampsia measurements with indium-113m and a computer-linked gamma camera. Clin Hypertens B 1:105–117Google Scholar
  32. Magness RR (1991) National Institutes of Health symposium. Endothelium-derived vasoactive substances and uterine blood vessels. Semin Perinatol 15:68–78Google Scholar
  33. Magness RR, Shaw CE, Phernetton TM, Zheng J, Bird IM (1997) Endothelial vasodilator production by uterine and systemic arteries II pregnancy effects on NO synthase expression. Am J Physiol Heart Circ Physiol 272:H1730–H1740CrossRefGoogle Scholar
  34. Maher J, Hunter AC, Mabley JG, Lippiat J, Allen MC (2013) Smooth muscle relaxation and activation of the large conductance Ca2+−activated K+ (BKCa) channel by novel oestrogens. Br J Pharmacol 169:1153–1165CrossRefGoogle Scholar
  35. Maurice DH, Crankshaw D, Haslam RJ (1991) Synergistic actions of nitrovasodilators and isoprenaline on rat aortic smooth muscle. Eur J Pharmacol 192:235–242CrossRefGoogle Scholar
  36. McGuire JJ, Ding H, Triggle CR (2001) Endothelium derived relaxing factors: a focus on endothelium-derived hyperpolarizing factor(s). Can J Physiol Pharmacol 79(6):443–470CrossRefGoogle Scholar
  37. McNeish AJ, Sandow SL, Neylon CB, Chen MX, Dora KA, Garland CJ (2006) Evidence for involvement of both IKCa and SKCa channels in hyperpolarizing responses of the rat middle cerebral artery. Stroke. 37:1277–1282CrossRefGoogle Scholar
  38. Morton JS, Jackson VM, Daly CJ, McGrath JC (2007) Endothelium dependent relaxation in rabbit genital resistance arteries is predominantly mediated by endothelial-derived hyperpolarizing factor in females and nitric oxide in males. J Urol 177:786–791CrossRefGoogle Scholar
  39. Nelson SH, Suresh MS (1988) Comparison of nitroprusside and hydralazine in isolated uterine arteries from pregnant and non-pregnant patients. Anesthesiology. 68:541–547CrossRefGoogle Scholar
  40. Nelson SH, Steinsland OD, Wang Y, Yallampalli C, Dong YL, Sanchez JM (2000) Increased nitric oxide synthase activity and expression in the human artery during pregnancy. Circ Res 87:406–411CrossRefGoogle Scholar
  41. Nelson SH, Steinsland OS, Johnson RL, Suresh MS, Gifford A, Ehardt JS (1995) Pregnancy-induced alterations of neurogenic constriction and dilation of the human uterine artery. Am J Physiol Heart Circ Physiol 268:H1694–H1701CrossRefGoogle Scholar
  42. Ni Y, Meyer M, Osol G (1997) Gestation increases nitric oxide-mediated vasodilation in rat uterine arteries. Am J Obstet Gynecol 176:856–864CrossRefGoogle Scholar
  43. Osol G, Barron C, Gokina N, Mandala M (2009) Inhibition of nitric oxide synthases abrogates pregnancy-induced uterine vascular expansive remodeling. J Vasc Res 46(5):478–486CrossRefGoogle Scholar
  44. Randall MD, Griffith TM (1991) Differential effects of L-arginine on the inhibition by NG-nitro-L-arginine methyl ester of basal and agonist-stimulated EDRF activity. Br J Pharmacol 104:743–749CrossRefGoogle Scholar
  45. Rosenfeld CR (1977) Distribution of cardiac output in ovine pregnancy. Am J Physiol Heart Circ Physiol 232:H231–H235CrossRefGoogle Scholar
  46. Rosenfeld CR, Cox BE, Roy T, Magness RR (1996) Nitric oxide contributes to estrogen-induced vasodilation of the ovine uterine circulation. J Clin Invest 98:2158–2166CrossRefGoogle Scholar
  47. Rosenfeld CR, Morris FH Jr, Makowski EL, Meschia G, Battaglia FC (1974) Circulatory changes in the reproductive tissues of ewes during pregnancy. Gynecol Investig 5:252–268CrossRefGoogle Scholar
  48. Scotland RS, Madhani M, Chauhan S, Moncada S, Andresen J, Nilsson H, Hobbs AJ, Ahluwalia A (2005) Investigation of vascular responses in endothelial nitric oxide synthase/cyclooxygenase-1 double-knockout mice: key role for endothelium-derived hyperpolarizing factor in the regulation of blood pressure in vivo. Circulation. 111(6):796–803CrossRefGoogle Scholar
  49. Senadheera S, Bertrand PP, Grayson TH, Leader L, Murphy TV, Sandow SL (2013) Pregnancy-induced remodelling and enhanced endothelium-derived hyperpolarization-type vasodilator activity in rat uterine radial artery: transient receptor potential vanilloid type 4 channels, caveolae and myoendothelial gap junctions. J Anat 223(6):677–686CrossRefGoogle Scholar
  50. Senadheera S, Kim Y, Grayson TH, Toemoe S, Kochukov MY, Abramowitz J, Housley GD, Bertrand RL, Chadha PS, Bertrand PP, Murphy TV, Tare M, Birnbaumer L, Marrelli SP, Sandow SL (2012) Transient receptor potential canonical type 3 channels facilitate endothelium-derived hyperpolarization mediated resistance artery vasodilator activity. Cardiovasc Res 95:439–447CrossRefGoogle Scholar
  51. Shimokawa H, Flavahan NA, Lorenz RR, Vanhoutte PM (1988) Prostacyclin releases endothelium-derived relaxing factor and potentiates its action in coronary arteries of the pig. Br J Pharmacol 95:1197–1203CrossRefGoogle Scholar
  52. Siddegowda YK, Leo MD, Kumar D, Hooda OK, Prakash VR, Mishra SK (2007) Influence of heat stress on the reactivity of isolated chicken carotid artery to vasoactive agents. Exp Physiol 92(6):1077–1086CrossRefGoogle Scholar
  53. Singh TU, Kathirvel K, Choudhury S, Garg SK, Mishra SK (2010) Eicosapentaenoic acid-induced endothelium-dependent and –independent relaxation of sheep pulmonary artery. Eur J Pharmacol 636(1–3):108–113CrossRefGoogle Scholar
  54. Sladek SM, Magness RR, Conrad KP (1997) Nitric oxide and pregnancy. Am J Phys Regul Integr Comp Phys 272:R441–R463Google Scholar
  55. Sorensen CM, Giese I, Braunstein TH, Holstein-Rathlou NH, Salomonsson M (2011) Closure of multiple types of K+ channels is necessary to induce changes in renal vascular resistance in vivo in rats. Pflugers Arch 462:655–667CrossRefGoogle Scholar
  56. Subramani J, Leo MD, Kathirvel K, Arunadevi R, Singh TU, Prakash VR, Mishra SK (2010) Essential role of nitric oxide in sepsis-induced impairment of endothelium-derived hyperpolarizing factor-mediated relaxation in rat pulmonary artery. Eur J Pharmacol 630(1–3):84–91CrossRefGoogle Scholar
  57. Sukumaran SV, Singh TU, Parida S, Reddy CEN, Thangamalai R, Kandasamy K, Singh V, Mishra SK (2013) TRPV4 channel activation leads to endothelium-dependent relaxation mediated by nitric oxide and endothelium-derived hyperpolarizing factor in rat pulmonary artery. Pharmacol Res 78:18–27CrossRefGoogle Scholar
  58. Tare M, Parkington HC, Coleman HA, Neild TO, Dusting GJ (1990) Hyperpolarization and relaxation of arterial smooth muscle caused by nitric oxide derived from the endothelium. Nature. 346:69–71CrossRefGoogle Scholar
  59. Vallance P, Collier J, Moncada S (1989) Effects of endothelium-derived nitric oxide on peripheral arteriolar tone in man. Lancet. 2:997–1000CrossRefGoogle Scholar
  60. Vanhoutte PM (1996) Endothelium-derived hyperpolarizing factor, vol XXI. Harwood academic publishers, Amsterdam, p 338Google Scholar
  61. Veerareddy S, Campbell ME, Williams SJ, Baker PN, Davidge ST (2004) Myogenic reactivity is enhanced in rat radial uterine arteries in a model of maternal under-nutrition. Am J Obstet Gynecol 191(1):334–339CrossRefGoogle Scholar
  62. Veerareddy S, Cooke CL, Baker PN, Davidge ST (2002) Vascular adaptations to pregnancy in mice: effects on myogenic tone. Am J Physiol Heart Circ Physiol 283:H2226–H2233CrossRefGoogle Scholar
  63. Wahab HA, El-Dina DS, Zain E, Mohamed MA, Youssefa AFM (2011) Uterine artery doppler and subendometrial blood flow in patients with unexplained recurrent miscarriage. Middle East Fer Soc J 16(3):209–214CrossRefGoogle Scholar
  64. Weiner C, Lui KZ, Thompson L, Herrig J, Chestnut D (1991) Effect of pregnancy on endothelium and smooth muscle: their role in reduced adrenergic sensitivity. Am J Physiol Heart Circ Physiol 261:H1275–H1283CrossRefGoogle Scholar
  65. Xiao D, Buchholz JN, Zhang L (2006) Pregnancy attenuates uterine artery pressure-dependent vascular tone: role of PKC/ERK pathway. Am J Physiol Heart Circ Physiol 290:H2337–H2343CrossRefGoogle Scholar
  66. Xiao D, Huang X, Yang S, Longo LD, Zhang L (2010) Pregnancy downregulates actin polymerization and pressure-dependent myogenic tone in ovine uterine arteries. Hypertension. 56(5):1009–1015CrossRefGoogle Scholar
  67. Xiao D, Pearce W, Zhang L (2001) Pregnancy enhances endothelium-dependent relaxation of ovine uterine artery: role of NO and intracellular Ca2+. Am J Physiol Heart Circ Physiol 281:H183–H190CrossRefGoogle Scholar
  68. Zamudio S, Palmer SK, Dahms TE, Berman JC, Young DA, Moore LG (1995) Alterations in uteroplacental blood flow precede hypertension in pre-eclampsia at high altitude. J Appl Physiol 79(1):15–22CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Udayraj P. Nakade
    • 1
  • Abhishek Sharma
    • 1
  • Priyambada Kumari
    • 1
  • Shirish Bhatiya
    • 1
  • Sooraj V. Nair
    • 1
  • K. N Karikaran
    • 1
  • Vipin Sharma
    • 1
  • Soumen Choudhury
    • 1
  • Satish Kumar Garg
    • 1
    Email author
  1. 1.Smooth Muscle and Molecular Pharmacology Laboratory, Department of Veterinary Pharmacology and Toxicology, College of Veterinary Science and Animal HusbandryU.P. Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go-Anusandhan Sansthan (DUVASU)MathuraIndia

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