WITHDRAWN: Differential involvement of L- and T-type Ca2+ channels, store-operated calcium channel (TRPC) and Rho-kinase signaling pathway(s) in PGF2α-induced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

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.

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.

Endocannabinoid-mediated modulation of Gq protein-coupled receptor mediates vascular hyporeactivity to nor-adrenaline during polymicrobial sepsis.

BACKGROUND: Endocannabinoids level are reported to increase in sepsis, however, the role of vascular cannabinoid receptor-1 (CB1R) in sepsis-induced vascular hyporeactivity is yet to be unravelled. METHODS: Polymicrobial sepsis was induced by caecal ligation and puncture in mice. Isometric tension in isolated aortic rings during early (6 h) and late (20 h) phases of sepsis was recorded and expression of mRNA of monoacylglycerol lipase (MAGL) and cannabinoid receptor-1 (CB1R) was investigated. RESULTS: Sepsis significantly (p < 0.001) reduced the mean survival time in mice along with increase in bacterial load in blood and peritoneal lavage. Compared to Sham-operated (SO) mice, vascular reactivity to nor-adrenaline (NA) was significantly (p < 0.05) attenuated in both early and late phases of sepsis. NA-induced vasoconstriction was significantly (p < 0.05) potentiated by inhibition of diacylglycerol lipase (DAGL) and attenuated by inhibition of MAGL in SO mice. Pre-incubation with KT 109, a DAGL inhibitor, significantly (p < 0.05) improved the vascular hypo-reactivity to NA during both the phases of sepsis. mRNA expression of MAGL in aorta was significantly (p < 0.05) attenuated during both the phases of sepsis. But in the presence of AM 251, specific antagonist of CB1R, vascular reactivity to NA was significantly (p < 0.05) restored along with significant (p < 0.05) increase in mRNA expression of CB1R in aortic rings from both early and late phases of septic mice. CONCLUSION: 2-AG regulates vascular response to NA and increased aortic expression of CB1R is responsible for vascular hyporeactivity to NA in sepsis, and in vitro inhibition of this receptor by AM 251 restored the vascular reactivity.

Trypanosoma evansi induces detrimental immuno-catabolic alterations and condition like type-2 diabetes in buffaloes.

The present study aimed to investigate the perturbations in immuno-metabolic and redox status of buffaloes with trypanosomosis. Thirteen buffaloes suffering from clinical trypanosomosis and eight apparently healthy buffaloes were included in the present study. Buffaloes with trypanosomosis found to have markedly elevated levels of interleukin-10 (IL-10), nonesterified fatty acids (NEFA) and beta-hydroxybutyrate (BHBA) in comparison with healthy controls. Whereas, total antioxidant capacity (TAC) and haemoglobin levels of buffaloes with trypanosomosis were significantly lower than the healthy controls. Remarkable elevation in malondialdehyde (MDA) and protein carbonyls (PC) levels were also observed in the diseased buffaloes. Moreover, buffaloes with trypanosomosis were found to have markedly elevated levels of serum glucose, total proteins, globulins, urea and aspartate aminotransferase (AST) and markedly lowered levels of serum calcium, total cholesterol levels and albumin/globulin (A/G) ratio as compared to the controls. Findings of our study evidently suggest that Trypanosoma evansi induces remarkable immunosuppressive and pro-oxidative status with an increased catabolic activity and hyperglycemic condition like type-2 diabetes in naturally infected buffaloes. Therefore, immuno-metabolic and pro-oxidative predicaments should be addressed by the veterinary clinician while managing the clinical cases of trypanosomosis in buffaloes.

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.

Functional involvement of protein kinase C, Rho-kinase and TRPC3 decreases while PLC increases with advancement of pregnancy in mediating oxytocin-induced myometrial contractions in water buffaloes (Bubalus bubalis).

Present study unravels the involvement of different calcium signaling pathways in oxytocin-induced contractions in myometrium of non-pregnant and pregnant buffaloes during early and mid-pregnancy stages. Uteri of pregnant animals were more sensitive than of non-pregnant buffaloes. Phasic contractions and frequency of contraction significantly increased with advancement of pregnancy, while tonic contractions non-significantly and amplitude significantly decreased from six months pregnancy onward. Oxytocin produced concentration-dependent-contraction on isolated myometrial strips of pregnant and non-pregnant buffaloes and the dose response curves (DRCs) of oxytocin were significantly (P < 0.05) shifted to right in the presence of nifedipine (1 µM), in Ca2+-free Ringer Locke solution (RLS), ruthenium red (30 µM), ruthenium red + nifedipine, cyclopiazonic acid (CPA; Ca2+ free RLS as well as RLS), CPA (10 µM)+nifedipine, U-73122 (1 µM) + nifedipine and SKF96365 (25 µM) on uteri of non-pregnant and pregnant (early and mid) animals. The DRCs were also significantly shifted towards right in the presence of Y-27632 (10 µM), GF109203X (5 µM) and Pyr3 (10 µM) on uteri of non-pregnant and early pregnancy stage buffaloes while only in the presence of U-73122 (1 µM) on uteri of mid-pregnancy stage buffaloes. Our finding suggest that and L-type Ca2+ channels, IP3-RyR-gated, and store-operated calcium channels including transient receptor potential channel (TRPC) pathways play significant role in mediating oxytocin-induced contractions in myometrium of pregnant and non-pregnant buffaloes. SERCA plays major role only during early-pregnancy while functional role of protein kinase C (PKC), Rho-kinase and TRPC3 pathways decreased and role of G-protein coupled receptor-phospholipase C (GPCR-PLC) pathway increased with advancement of pregnancy.

Lead Modulates Calcium Entry and Beta-Adrenoceptors Signaling to Produce Myometrial Relaxation in Rats.

Modulation of myometrial spontaneity by lead acetate trihydrate (Pb) and its regulatory pathways were studied in estrogenized rats. Isometric tension in myometrial strips under a resting tension of 1 g was measured using data acquisition system-based physiograph and Lab Chart Pro v7.3.7 software. Lead produced a dose-dependent inhibitory effect on rat myometrium with a major effect on phasic contractions compared to tonic contractions along with a reduction in both amplitude and frequency of contraction. Lead (3 µM) significantly (p < 0.05) reduced CaCl2, and 80 mM KDS induced contractile response while potentiated the relaxant effect of phenylephrine. Based on our findings, it may be inferred that lead blocks calcium entry through VDCC and/or stimulates ß-adrenoceptors adenylyl cyclase-C-AMP pathway to produce inhibitory effect on rat myometrium.

Extra and intracellular calcium signaling pathway(s) differentially regulate histamine-induced myometrial contractions during early and mid-pregnancy stages in buffaloes (Bubalus bubalis).

This study examines the differential role of calcium signaling pathway(s) in histamine-induced uterotonic action during early and mid-pregnancy stages in buffaloes. Compared to mid pregnancy, tonic contraction, amplitude and mean-integral tension were significantly increased by histamine to produce myometrial contraction during early pregnancy with small effects on phasic contraction and frequency. Although uterotonic action of histamine during both stages of pregnancy is sensitive to nifedipine (a L-type Ca2+ channels blocker) and NNC55-0396 (T-type Ca2+ channels blocker), the role of extracellular calcium seems to be more significant during mid-pregnancy as in this stage histamine produced only 9.38±0.96% contraction in Ca2+ free-RLS compared to 21.60±1.45% in uteri of early pregnancy stage. Intracellular calcium plays major role in histamine-induced myometrial contraction during early pregnancy as compared to mid pregnancy, as in the presence of cyclopiazonic acid (CPA) Ca2+-free RLS, histamine produced significantly higher contraction in myometrial strips of early-pregancy in comparison to mid-pregnancy (10.59±1.58% and 3.13±0.46%, respectively). In the presence of U-73122, the DRC of histamine was significantly shifted towards right with decrease in maximal effect (Emax) only in early pregnancy suggesting the predominant role of phospholipase-C (PL-C) in this stage of pregnancy.

Functional and molecular characterization of voltage gated sodium channel Nav 1.8 in bull spermatozoa.

The aim of our study was to characterize the voltage gated sodium channel Nav 1.8 in bull spermatozoa. Forty ejaculates were collected from four Hariana bulls and semen samples were pooled in view of the nonsignificant variations between different ejaculates. Functional characterization was undertaken using A-803467, a selective blocker of Nav1.8, and veratridine as an opener of the voltage gated sodium channels while molecular characterization was done using western blotting and indirect immunofluorescence assays. In vitro capacitation was induced using heparin, and to study the functional involvement of Nav 1.8 in regulation of capacitation induced hyper sperm motility, A-803467 was used. Selective blocking of NaV 1.8 by A-803467 at 6 and 8 µM concentration significantly (P < 0.05) decreased the forward progressive sperm motility in a time-dependent manner, while, blocking at higher concentrations (10 and 15 µM) resulted in fast forward motility in spermatozoa after 2 h of incubation and it was observed up to 3 h. Treatment of sperm cells with veratridine (6, 8, 10, 15, 20 µM) resulted in concentration- and time-dependent increase in forward progressive sperm motility and it persisted up to 4 h. However, hyperactive motility was induced by veratridine at higher concentrations (10 and 15 µM) after 2 h of incubation. In vitro capacitated spermatozoa treated with A-803467 revealed significant (P < 0.05) reduction in forward progressive motility after 2 h of incubation. Both A-803467 and veratridine altered the percentage of spermatozoa showing high mitochondrial transmembrane potential in concentration- and time-dependent manner. High concentrations (10 and 15 µM) of A-803467 and veratridine resulted in bent neck condition in spermatozoa along with significant (P < 0.05) reduction in membrane integrity (HOST negative). Immunoblot revealed the presence of a single protein band of 260 kDa molecular weight along with positive immunoreactivity (IR) in head, neck, middle piece and tail of the spermatozoa. Strongest IR was observed in the neck and middle piece whereas weak IR was observed in tail and acrosomal region of the spermatozoa. Results of our present study evidently revealed the presence of voltage gated sodium channel Nav1.8 in bull spermatozoa and its functional involvement in regulation of spermatozoa dynamics in terms of motility, membrane integrity, acrosome integrity, capacitation and mitochondrial transmembrane potential. Further studies are warranted to unravel their mechanistic pathways and/or their interaction with other ion channels in regulating sperm dynamics.

Ameliorative Potential of Prepartal Trace Mineral and Vitamin Supplementation on Parturition-Induced Redox Balance and Myeloperoxidase Activity of Periparturient Sahiwal Cows.

Twelve apparently healthy multiparous parturient Sahiwal cows were allocated into two groups having six cows in each one. Six cows were supplemented with antioxidant mixture (mixture containing Cu, Mn, Cr, Zn, and vitamins A and D3) daily from 21 days before parturition till the day relative to calving. Whereas, remaining non-supplemented six cows were kept as the control group. Blood samples were obtained five times: at enrolment (21 days pre-partum), and again at days 0, +7, +14, and +21 relative to calving. In the non-supplemented control group, serum total antioxidant capacity (TAC) was significantly lower at days 0, +7, and +14 as compared to their own day -21 values. Likewise, significantly lower myeloperoxidase (MPO) activities were also exhibited by these cows at days 0 and +7. Conversely, serum malondialdehyde (MDA) and protein carbonyl (PC) levels were significantly higher in these cows at days 0, +7, +14, and +21. However, significant alterations in TAC content among the studied sampling days were not recorded in antioxidants supplemented group. Moreover, TAC content and MPO activities of supplemented group were significantly higher at days 0, +7, and +14 when compared with that of the non-supplemented control group. However, MDA and PC contents of supplemented group were significantly lower at days 0, +7, +14, and +21 as compared to that of the non-supplemented control group. In conclusion, periparturient Sahiwal cows experience substantial oxidative and immunological dents which can be potentially ameliorated by prepartal trace mineral and vitamin supplementation.

Potential association of reduced cholinesterase activity with Trypanosoma evansi pathogenesis in buffaloes.

The present study aimed to investigate the association of cholinesterase activity with trypanosomosis in buffaloes. Thirty-three clinical cases of trypanosomosis in water buffaloes, found positive for trypomastigotes of T. evansi on blood smear examination, were divided into two groups based on clinical manifestations. Twenty diseased buffaloes revealing only common clinical signs were allocated to Group I, while the remaining 13 buffaloes showing common clinical manifestations along with neurological disturbances were allocated to Group II. Twelve clinically healthy buffaloes, free from any haemoprotozoa infection, were kept as healthy control (Group III). Blood samples were collected from buffaloes of all three groups to determine serum cholinesterase activity. Compared to buffaloes of healthy control group, cholinesterase activity in T. evansi-infected buffaloes of Group I and II was significantly (P<0.001) lower. However, no significant difference was observed in cholinesterase activity between the T. evansi-infected buffaloes exhibiting neurological disorders and no neurological disorders. Summing up, reduced cholinesterase activity seems to be associated with the pathogenesis of natural T. evansi infection and its clinical manifestations in buffaloes possibly by evading immune response. Further studies are warranted on association of cholinesterase activity in T. evansi-infected buffaloes with neurological disorders.

Calcium influx and release mechanism(s) in histamine-induced myometrial contraction in buffaloes.

The present study was undertaken to characterize the presence of histamine H1R using molecular biology tools and unravel the influx and release mechanism(s) involved in calcium signalling cascades in histamine-induced myometrial contraction in buffaloes. The presence of H1R mRNA transcript and immunoreactive membrane protein in buffalo myometrium was confirmed by RT-PCR and Western blot analysis. Further, histamine produced concentration-dependent (1nM-10µM) contraction in buffalo myometrium with a potency of 7.13±0.11. When myometrial strips were pre-incubated either with Ca(2+) free solution or with nifedipine, a L-type Ca(2+) channel blocker, dose response curve (DRC) of histamine was significantly (P<0.05) shifted towards right with decline in maximal contraction (Emax). Reduction in Emax of histamine in the presence of nifedipine (55.75±3.10%) was significantly (P<0.001) greater than that in the presence of ruthenium red (93.61±3.43%), a blocker of IP3-gated and RyR-sensitive Ca(2+) channels. Moreover, histamine produced only 26.87±1.99% of the maximum contraction in the presence of both nifedipine and CPA (blocker of sarco-endoplasmic reticulum Ca(2+)-ATPase). Interestingly, following concurrent exposure to U-73122 (a PL-C inhibitor) and nifedipine, the DRC of histamine was significantly (P<0.05) shifted towards left with increase in maximal contraction (126.30±3.36%). Our findings in buffalo uterus thus suggest that influx of extracellular calcium plays a major role in histamine-induced myometrial contraction, while release of intracellular calcium through calcium-release channels of sarcoplasmic reticulum has a minor role. A possible involvement of non-selective cation channels in histamine-induced myometrial contraction cannot be ruled out, and therefore requires further investigations.