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

Abstract

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.

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Funding

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.

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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.

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Correspondence to Satish Kumar Garg.

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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).

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Nakade, U.P., Sharma, A., Kumari, P. et al. Functional and molecular characterization of endothelium-dependent and endothelium-independent relaxant pathways in uterine artery of non-pregnant buffaloes. Naunyn-Schmiedeberg's Arch Pharmacol 393, 225–241 (2020). https://doi.org/10.1007/s00210-019-01726-y

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Keywords

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