Methionine supply during the peripartum period and early lactation alter immunometabolic gene expression in cytological smear and endometrial tissue of holstein cows.

The objective of the present study was to evaluate the effect of feeding rumen-protected methionine (RPM) during the peripartal period and early lactation on mRNA gene expression profiles of uterine cytological smear and endometrial samples of Holstein cows (n = 20). Treatments consisted of a supplementation with RPM [MET; n = 11; RPM at a rate of 0.08 % of DM: Lys:Met = 2.8:1, (Smartamine® M Adisseo, Alpharetta, GA, USA)] and no supplementation (CON; n = 9; Lys:Met = 3.5:1). Uterine cytology smears and endometrial samples were collected at 15, 30, and 73 days in milk (DIM) and analyzed for expression of genes related with metabolism, inflammation, and methionine metabolism. Regarding the cytological smear samples, RPM supplementation tended to increase mRNA expression of methionine adenosyltransferase 1 alpha (MAT1A) and increased the mRNA expression of fibroblast growth factor 7 (FGF7), with an effect of time for the latter. On the other hand, RPM decreased mRNA expression for glucose transporter 4 (GLUT4), interleukin 1 beta (IL-1ß), interleukin 6 (IL-6), interleukin 8 (IL-8), prostaglandin E synthase 3 (PTGES3), translocator protein 18 kDa (TSPO), mucin 1 (MUC1) and superoxide dismutase (SOD1) in cytological smear samples. There was an effect of time for all variables except MAT1A, with decreasing expression over time. There was a TRT × TIME interaction for GLUT4 mRNA expression, with higher GLUT4 mRNA expression for cows fed CON than for cows fed RPM at time 15 and a tendency to higher expression for cows fed CON on time 30 when compared with cows fed RPM. For uterine tissue samples, feeding RPM increased the mRNA expression of lecithin-cholesterol acyltransferase (LCAT), S-adenosyl-l-homocysteine hydrolase (SAAH), FGF7, GLUT4, and apolipoproteins 3 (APOL3), with an effect of time for APOL3 where its expression increased over time. There was a tendency for cows fed RPM to have decreased IL1ß mRNA expression. In conclusion, feeding RPM during transition period and early lactation is beneficial for uterine immune response and metabolism in early lactation as indicated by the favorable expressions of genes affecting the uterine immunometabolism during such a challenging period.

Improved uterine immune mediators in Holstein cows supplemented with rumen-protected methionine and discovery of neutrophil extracellular traps (NET).

During the transition from prepartum to early lactation, dairy cows often experience negative energy balance (NEB) that may result in reproductive stress and decreased fertility. The objective of this study was to observe the effects of rumen-protected methionine (RPM) on plasma amino acid concentrations, uterine cytology, immunohistochemistry (IHC) of glutathione peroxidase 1 (GPX) and superoxide dismutase 1 (SOD), and to confirm neutrophil extracellular trap (NET) formation. Multiparous Holstein cows (n = 20) were randomly assigned to two treatments starting at 21 d before calving until 73 days in milk (DIM). Treatments were: CON (n = 9, no supplementation, TMR with a Lys:Met = 3.5:1) and MET (n = 11, TMR + Smartamine® M with a Lys:Met = 2.8:1). Uterine endometrial biopsies, uterine cytology, and blood samples from the coccygeal artery or vein were collected at 15, 30, and 73 DIM. Blood plasma samples were analyzed for amino acids and metabolites. Uterine biopsies were analyzed for NET formation, neutrophil numbers, as well as GPX and SOD by IHC. Additionally, uterine cytology was analyzed for polymorphonuclear neutrophil (PMN) to epithelial cell percentage. Cows in CON had lower methionine plasma concentrations (18.05 ±â€¯2.0 µM) than cows in MET (30.39 ±â€¯1.6 µM). Cows in CON had greater cystine plasma concentrations (3.62 ±â€¯0.3 µM) than cows in MET (2.8 ±â€¯0.3 µM). No treatment differences were observed for SOD or GPX in the endometrium. Cows in CON tended to have a high score for positively immunolabeled GPX cells at 15 DIM than cows in MET. No treatment differences were observed for the percentage of PMN in uterine cytology, number of neutrophils, or extent of NET formation in the endometrium. A treatment by time interaction was observed for PMN percentage and the number of neutrophils: cows in MET tended to have greater PMN percentages than cows in CON at 15 DIM which decreased for subsequent days and cows in MET had greater neutrophil numbers in the endometrium at 30 DIM than cows in CON. In conclusion, dietary supplementation of RPM altered plasma amino acid concentrations and increased neutrophil infiltration in the postpartum period, suggesting improved uterine immunity.

Location of release sites and calcium-activated chloride channels relative to calcium channels at the photoreceptor ribbon synapse.

Vesicle release from photoreceptor ribbon synapses is regulated by L-type Ca(2+) channels, which are in turn regulated by Cl(-) moving through calcium-activated chloride [Cl(Ca)] channels. We assessed the proximity of Ca(2+) channels to release sites and Cl(Ca) channels in synaptic terminals of salamander photoreceptors by comparing fast (BAPTA) and slow (EGTA) intracellular Ca(2+) buffers. BAPTA did not fully block synaptic release, indicating some release sites are <100 nm from Ca(2+) channels. Comparing Cl(Ca) currents with predicted Ca(2+) diffusion profiles suggested that Cl(Ca) and Ca(2+) channels average a few hundred nanometers apart, but the inability of BAPTA to block Cl(Ca) currents completely suggested some channels are much closer together. Diffuse immunolabeling of terminals with an antibody to the putative Cl(Ca) channel TMEM16A supports the idea that Cl(Ca) channels are dispersed throughout the presynaptic terminal, in contrast with clustering of Ca(2+) channels near ribbons. Cl(Ca) currents evoked by intracellular calcium ion concentration ([Ca(2+)](i)) elevation through flash photolysis of DM-nitrophen exhibited EC(50) values of 556 and 377 nM with Hill slopes of 1.8 and 2.4 in rods and cones, respectively. These relationships were used to estimate average submembrane [Ca(2+)](i) in photoreceptor terminals. Consistent with control of exocytosis by [Ca(2+)] nanodomains near Ca(2+) channels, average submembrane [Ca(2+)](i) remained below the vesicle release threshold (∼ 400 nM) over much of the physiological voltage range for cones. Positioning Ca(2+) channels near release sites may improve fidelity in converting voltage changes to synaptic release. A diffuse distribution of Cl(Ca) channels may allow Ca(2+) influx at one site to influence relatively distant Ca(2+) channels.

Insulin inhibits voltage-dependent calcium influx into rod photoreceptors.

Insulin inhibits the ERG b-wave and modulates L-type calcium currents (I(Ca)) in various preparations. We therefore examined insulin's effects on I(Ca) and depolarization-evoked [Ca2+]i increases in rod photoreceptors. Insulin inhibited I(Ca) and caused a dose-dependent reduction in the depolarization-evoked Ca2+ influx with an EC50 of 2.1 nM. Tyrosine kinase inhibitors, lavendustin A (100 nM) and genistein (10 microM), prevented insulin from reducing the depolarization-evoked Ca2+ increase in rods. Their less active analogues, lavendustin B and daidzein, had similar effects. An insulin receptor-specific tyrosine kinase inhibitor, HNMPA-(AM)3 (50 microM), prevented insulin (30 nM) from reducing the depolarization-evoked Ca2+ increase in rods. The results suggest that insulin inhibits Ca2+ influx through voltage-dependent I(Ca) in rod photoreceptors via tyrosine kinase activity.

Differential modulation of rod and cone calcium currents in tiger salamander retina by D2 dopamine receptors and cAMP.

Synaptic transmission from vertebrate photoreceptors involves activation of L-type calcium currents (ICa). Dopamine is an important circadian neuromodulator in the retina and photoreceptors possess D2 dopamine receptors. We examined modulation of ICa by dopamine and cAMP in retinal slices and isolated cells of larval tiger salamander. Results show that dopamine and a D2 agonist, quinpirole, enhanced ICa in rods and red-, blue- and UV-sensitive small single cones but inhibited ICa in red-sensitive large single cones. A D1 agonist, SKF-38393, was without effect. Quinpirole effects were blocked by pertussis toxin (PTx) pretreatment indicating involvement of PTx-sensitive G-proteins. Like dopamine, inhibition of cAMP-dependent protein kinase (PKA) by Rp-cAMPS enhanced ICa in rods and small single cones, but inhibited ICa in large single cones. In contrast, forskolin and Sp-cAMPS, which stimulate PKA, inhibited ICa in rods and small single cones but enhanced ICa in large single cones. Sp-cAMPS also occluded effects of quinpirole. These results suggest that D2 receptors modulate ICa via inhibition of cAMP. Differences among the responses of photoreceptors to cAMP are consistent with the possibility that small single cones and rods may possess different Ca2+ channel subtypes than large single cones. The results with dopamine and quinpirole showing inhibition of ICa in large single cones and enhancement of rod ICa were unexpected because previous studies have shown that dopamine suppresses rod inputs and enhances cone inputs into second-order neurons. The present results therefore indicate that the dopaminergic enhancement of cone inputs does not arise from modulation of photoreceptor ICa.

Anion modulation of calcium current voltage dependence and amplitude in salamander rods.

Hofmeister anions were used to investigate the ability of Cl(-) replacement to produce inhibition and a hyperpolarizing activation shift in L-type Ca(2+) currents (I(Ca)) of rod photoreceptors. Inhibition of I(Ca) largely followed the Hofmeister sequence: Cl(-)=Br(-)

Involvement of cytoplasmic calcium and protein kinases in the regulation of atrial natriuretic factor secretion by contraction rate and endothelin.

To characterize the effects of the cellular events associated with contraction on atrial natriuretic factor (ANF) secretion, primary neonatal rat atrial myocytes were electrically paced to contract while being monitored for ANF release, cytoplasmic calcium, phosphoinositide hydrolysis, and protein kinase C activation. Similar measurements were also carried out in the presence of endothelin-1 (ET) for comparison of contraction-related and hormone-stimulated ANF secretion. Pacing (6-8 Hz) immediately increased ANF secretion by 3-5-fold and the time-averaged cytoplasmic calcium concentration (as monitored with indo-1 fluorescence) varied with pace frequency in a similar manner, suggesting that cytoplasmic calcium may play a key role in pace-induced ANF secretion. Furthermore, nifedipine and ryanodine, which inhibited the contractile calcium transients, inhibited pace-induced ANF release, whereas Bay K 8644 increased both the calcium transients and ANF secretion. Pace-induced ANF release was also completely inhibited by KN-62, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMK) but was not inhibited by chelerythrine, a protein kinase C-selective inhibitor. Pace-induced ANF release averaged 40% of that elicited by ET which is known to require both PKC and CaMK for maximal effects on ANF secretion. The effects of pacing and ET on ANF secretion were approximately additive. In contrast to pacing, ET strongly stimulated phosphoinositide hydrolysis, activated PKC, and did not increase cytoplasmic calcium. Thus, regulation of ANF secretion by contraction rate depends primarily on the contractile calcium transients and CaMK and is independent of PKC.