Calcium signaling cascades differentially regulate PGF2α-induced myometrial contractions in water buffaloes (Bubalus bubalis).

This study unravels the differential involvement of calcium signaling pathway(s) in PGF2α-induced contractions in myometrium of nonpregnant (NP) and pregnant buffaloes. Compared to the myometrium of pregnant animals, myometrium of NP buffaloes was more sensitive to PGF2α as manifested by changes in mean integral tension (MIT) and tonicity. In the presence of nifedipine, myometrial contraction to PGF2α was significantly attenuated in both NP and pregnant uteri; however, mibefradil and NNC 55-0396 produced inhibitory effects only in uterus of pregnant animals, thus suggesting the role of extracellular Ca2+ influx through nifedipine-sensitive L-type Ca2+-channels both in NP and pregnant, but T-type Ca2+ channels seem to play a role only during pregnancy. Entry of extracellular Ca2+ is triggered by enhanced functional involvement of Pyr3-sensitive TRPC3 channels and Rho-kinase pathways as evidenced by a significant rightward shift of the concentration-response curve of PGF2α in the presence of Pyr3 and Y-27632 in NP myometrium. But significant down-expressions of TRPC3 and Rho-A proteins during pregnancy apparently facilitate uterine quiescence. In the presence of Ca2+-free solution and cyclopiazonic acid (SERCA blocker), feeble contraction to PGF2α was observed in both NP and pregnant myometrium which suggests minor role of intracellular source of Ca2+ in mediating PGF2α-induced contractions in these tissues.

Eucalyptus robusta leaves methanolic extract suppresses inflammatory mediators by specifically targeting TLR4/TLR9, MPO, COX2, iNOS and inflammatory cytokines in experimentally-induced endometritis in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Bacterial endometritis is one of the major causes of reproductive disorders including infertility in farm animals. Antibiotics are generally used for treatment of such disorders but now a days residues of antibiotics are of great public health concern, therefore, phytoremediation is being considered as an alternative to use of antibiotics. AIM OF THE STUDY: Present study was undertaken to investigate the efficacy of Eucalyptus robusta leaves methanolic extract against endometritis along with the possible mechanism of action especially targeting inflammatory biomarkers. MATERIALS AND METHODS: Bacterial endometritis was produced using clinical isolates of E. coli and Staphyloccocus aureus from bovines (cows and buffaloes) endometritis cases. After seven days of inoculation of the mixed bacterial culture, endometritis was confirmed based on the presence of visible pus and edema, thinning of endometrial lining and presence of large number of polymorphonuclear cells and bacterial load in uterine flushing. Female Wistar rats were divided in to five groups namely control, sham-operated, endometritis, endometritis plus Eucalyptus leaves extract and endometritis plus cefixime. Serum specific inflammatory biomarkers (interleukin-1ß, interleukin-10, tumor necrosis factor-α, intercellular adhesion molecule-1, serum amyloid A) and myleoperoxidase, toll like receptors-4 and -9, inducible nitric oxide synthase, nitric oxide, cyclooxygenase 1 and 2 were estimated in uterine tissues using ELISA kits. RESULTS: Interleukin-10, serum amyloid A, myleoperoxidase, toll like receptors-4 and-9, cyclooxygenase-2, inducible nitric oxide synthase and nitric oxide were significantly increased while non significant increase in interleukin-1ß, cycloxygenase-1 and intercellular adhesion molecule-1 were observed but level of tumor necrosis factor-α was found decreased in rats of endometritis group. Histopathological lesions in uterus showed efficient induction of endometritis by presence of inflammatory cells which are lessened effectively after treatment with Eucalyptus leaves extract. Eucalyptus robusta leaves extract produced curative and protective effect against endometritis and results were comparable to or even better than cefixime. CONCLUSIONS: Eucalyptus robusta leaves extract possess promising antibacterial activity and efficacy against experimental endometritis and, therefore, holds promising potential for development of effective formulation for treatment of endometritis in animals.

ATP-sensitive and maxi potassium channels regulate BRL 37344-induced tocolysis in buffaloes-an in vitro study.

Cellular coupling of beta3-adrenoceptors (ß3-ADR) to potassium channels in myometrium is largely unknown. In vitro study was undertaken to unravel the presence of ß3-adrenergic receptors (ADR) and the role of K+-channels in mediating ß3-ADR-induced relaxation in isolated myometrial strips from cyclic non-pregnant water buffaloes. Isometric tension was recorded in isolated myometrial strips using data acquisition system based physiograph. Compared to SR 59230A, BRL 37344 was found to be more potent in inducing ß3-dependent myometrial relaxation which was significantly (p < 0.05) inhibited in the presence of ß3 antagonist, SAR 150640. The immunoreactive protein to ß3-ADR was also detected in membrane fraction of myometrial protein. Further, incubation with BRL 37344 (10 µM) significantly (p < 0.05) increased c-AMP accumulation (37.58 ± 9.52 pmol/mg protein; n = 4) in the myometrial strips compared to basal c-AMP level (16.85 ± 3.87 pmol/mg protein; n = 4). The concentration response curves (CRC) of BRL 37344 were significantly (p < 0.05) shifted towards right in the presence of KATP channels specific blocker, glibenclamide (10 µM) and maxi K+-channels (BKCa) specific blocker, iberiotoxin (100 nM), with decrease in both efficacy and potency as compared to control. However, 4-aminopyridine (4-AP), a specific blocker of the voltage gated K+-channels (Kv), failed to alter the CRC of BRL 37344. Existence of immunoreactive protein to Kir6.1, α-subunit of BKCa and Kv1.1 channels were also detected in the membrane fraction of myometrial protein. Based on the above findings, it can be concluded that BRL 37344 is a potent stimulator of ß3-adrenoceptors in buffalo myometrium and besides mediating their effect through rise in c-AMP, they are coupled to KATP and BKCa channels in inducing tocolytic effects.

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.

WITHDRAWN: Differential involvement of L- and T-type Ca2+ channels, store-operated calcium channel (TRPC) and Rho-kinase signaling pathway(s) in PGF2α-induced contractions in myometrium of non-pregnant and pregnant buffaloes (Bubalus bubalis).

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