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

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.

Dose-Dependent Differential Effects of In Vivo Exposure of Cadmium on Myometrial Activity in Rats: Involvement of VDCC and Ca2+-Mimicking Pathways.

Present study was undertaken to study the effect of 28-days exposure of female adult rats to cadmium (Cd) in drinking water @ 3, 10 and 30 parts per million (ppm) on myometrial responsiveness to different spasmogens and unravel the possible mechanism of alterations in myometrial activity. Cadmium and Ca2+ levels in blood and uterus were measured by atomic absorption spectroscopy while isometric tension in myometrial strips was measured using data acquisition system-based physiograph. Dose-dependent increase in levels of cadmium was observed in both blood and uterus while calcium was increased only in the uterus as compared to those in control. Significant increase in absolute tension and mean integral tension along with non-significant increase in frequency of myometrial contraction was observed in rats of Cd-treated groups. As compared to the control, cadmium decreased and increased the effects of calcium chloride, 80 mM KCl, histamine (0.1 µM) and oxytocin (10-2 IU/ml) in lower-dose (3 ppm) and higher-dose groups (10 and 30 ppm), respectively. Cadmium potentiated and inhibited the relaxant response to phenylephrine in myometrium of rats at lower-dose (3 ppm) and highest-dose (30 ppm) Cd-treated groups, respectively. Results of our study revealed that Cd accumulates in the myometrium of rats and alters its responsiveness to oxytocin, histamine, 80 mM KCl, calcium chloride and phenylephrine, and these effects are differentially mediated depending on levels of exposure possibly through voltage-dependent calcium channel (VDCC) and Ca2+-mimicking pathways.

The functional and molecular studies on involvement of hydrogen sulphide in myometrial activity of non-pregnant buffaloes (Bubalus bubalis).

BACKGROUND: Hydrogen sulphide (H2S), a member of the gasotransmitters family, is known to play patho-physiological role in different body systems including during pregnancy. But its involvement in myometrial spontaneity and associated signalling pathways in uterus in non-pregnant animals is yet to be studied. Present study describes the effect of L-cysteine, an endogenous H2S donor, on isolated myometrial strips of non-pregnant buffaloes and the underlying signaling mechanism(s). RESULTS: L-cysteine (10 nM-30 mM) produced concentration-dependent contractile effect on buffalo myometrium which was extracellular Ca2+ and L-type calcium channels-dependent. Significant rightward shift of dose-response curve of L-cysteine was observed with significant decrease in maxima in the presence of amino-oxyacetic acid (AOAA; 100 µM) and d, l-propargylglycine (PAG; 100 µM), the specific blockers of cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE), respectively. Existence of CBS enzyme of 63 kDa and CSE of 45 kDa molecular weights was confirmed by western blot using specific antibodies and also by immunohistochemistry. CONCLUSIONS: Endogenous H2S along with its biosynthetic enzymes (CBS and CSE) is evidently present in uteri of non-pregnant buffaloes and it regulates spontaneity in uteri of non-pregnant buffaloes and this effect is dependent on extracellular Ca2+ influx through nifedipine-sensitive L-type calcium channels. Thus H2S-signalling pathway may be a potential target to alter the uterine activities in physiology and patho-physiolgical states.