Melatonin induces the expression of Nrf2-regulated antioxidant enzymes via PKC and Ca2+ influx activation in mouse pancreatic acinar cells.

The goal of this study was to evaluate the potential activation of the nuclear factor erythroid 2-related factor and the antioxidant-responsive element (Nrf2-ARE) signaling pathway in response to melatonin in isolated mouse pancreatic acinar cells. Changes in intracellular free Ca(2+) concentration were followed by fluorimetric analysis of fura-2-loaded cells. The activations of PKC and JNK were measured by Western blot analysis. Quantitative reverse transcription-polymerase chain reaction was employed to detect the expression of Nrf2-regulated antioxidant enzymes. Immunocytochemistry was employed to determine nuclear location of phosphorylated Nrf2, and the cellular redox state was monitored following MitoSOX Red-derived fluorescence. Our results show that stimulation of fura-2-loaded cells with melatonin (1 µM to 1 mM), in the presence of Ca(2+) in the extracellular medium, induced a slow and progressive increase of [Ca(2+)](c) toward a stable level. Melatonin did not inhibit the typical Ca(2+) response induced by CCK-8 (1 nM). When the cells were challenged with indoleamine in the absence of Ca(2+) in the extracellular solution (medium containing 0.5 mM EGTA) or in the presence of 1 mM LaCl(3), to inhibit Ca(2+) entry, we could not detect any change in [Ca(2+)](c). Nevertheless, CCK-8 (1 nM) was able to induce the typical mobilization of Ca(2+). When the cells were incubated with the PKC activator PMA (1 µM) in the presence of Ca(2+) in the extracellular medium, we observed a response similar to that noted when the cells were challenged with melatonin 100 µM. However, in the presence of Ro31-8220 (3 µM), a PKC inhibitor, stimulation of cells with melatonin failed to evoke changes in [Ca(2+)]c. Immunoblots, using an antibody specific for phospho-PKC, revealed that melatonin induces PKCα activation, either in the presence or in the absence of external Ca(2+). Melatonin induced the phosphorylation and nuclear translocation of the transcription factor Nrf2, and evoked a concentration-dependent increase in the expression of the antioxidant enzymes NAD(P)H-quinone oxidoreductase 1, catalytic subunit of glutamate-cysteine ligase, and heme oxygenase-1. Incubation of MitoSOX Red-loaded pancreatic acinar cells in the presence of 1 nM CCK-8 induced a statistically significant increase in dye-derived fluorescence, reflecting an increase in oxidation, that was abolished by pretreatment of cells with melatonin (100 µM) or PMA (1 µM). On the contrary, pretreatment with Ro31-8220 (3 µM) blocked the effect of melatonin on CCK-8-induced increase in oxidation. Finally, phosphorylation of JNK in the presence of CCK-8 or melatonin was also observed. We conclude that melatonin, via modulation of PKC and Ca(2+) signaling, could potentially stimulate the Nrf2-mediated antioxidant response in mouse pancreatic acinar cells.

Melatonin modulates Ca2+ mobilization and amylase release in response to cholecystokinin octapeptide in mouse pancreatic acinar cells

In the present work, we have evaluated the effect of an acute addition of melatonin on cholecystokinin octapeptide (CCK-8)-evoked Ca2+ signals and amylase secretion in mouse pancreatic acinar cells. For this purpose, freshly isolated mouse pancreatic acinar cells were loaded with fura-2 to study intracellular free Ca2+ concentration ([Ca2+]c). Amylase release and cell viability were studied employing colorimetric methods. Our results show that CCK-8 evoked a biphasic effect on amylase secretion, finding a maximum at a concentration of 0.1 nM and a reduction of secretion at higher concentrations. Pre-incubation of cells with melatonin (1 ìM–1 mM) significantly attenuated enzyme secretion in response to high concentrations of CCK-8. Stimulation of cells with 1 nM CCK-8 led to a transient increase in [Ca2+]c, followed by a decrease towards a constant level. In the presence of 1 mM melatonin, stimulation of cells with CCK-8 resulted in a smaller [Ca2+]c peak response, a faster rate of decay of [Ca2+]c and lower values for the steady state of [Ca2+]c, compared with the effect of CCK-8 alone. Melatonin also reduced the oscillatory pattern of Ca2+ mobilization evoked by a physiological concentration of CCK-8 (20 pM), and completely inhibited Ca2+mobilization induced by 10 pM CCK-8. On the other hand, Ca2+ entry from the extracellular space was not affected in the presence of melatonin. Finally, melatonin alone did not change cell viability. We conclude that melatonin, at concentrations higher than those found in blood, might regulate exocrine pancreatic function via modulation of Ca2+ signals (AU)

Whole linted cottonseed meal (Gossypium hirsutum L.) protein and fiber degradability in the rumen / Degradação da proteína e fibra do caroço de algodão integral (Gossypium hirsutum L.) no rúmen

Evaluation of in situ" degradability of DM, CP and ADF of whole linted cottonseed (WLC) when used up to 15% of the diet (dry matter basis), replacing cottonseed meal, was the main purpose of this experiment. Sorghum silage (SS) was the only roughage. Ruminal pH and rumen kinetics were also evaluated. Nine ruminal canulated steers were used in a3 x 3 change-over design to evaluate the following treatments: A = 0% WLC; B = 6.6% WLC; and C = 15.0% WLC. Sorghum silage contributed with 70% in all three treatments. DM degradability at 48h incubation time was statistically different (p 0.05) (A = 54.4%; B = 54.2% and C = 58.7%), as well as PB degradability at 12h (A = 40.3%; B = 47.7% and C = 53.1%) and ADF degradability at 48h (A = 40.3%; B = 41.2% and C = 45.6%). Ruminal volume, turn overtime and ruminal pH werent affected by the experimental diets. Substitution of WLC for cottonseed meal up to 15% diet increased degradability of DM, CP and ADF of WLC.

0 experimento teve como objetivo avaliar a degradabilidade in situ da matéria seca (MS), proteína bruta (PB) e fibra em detergente ácido (FDA) do caroço de algodão integral (CAI) em substituição ao farelo de algodão, empregando-se silagem de sorgo (SS), como único volumoso. Além disso, avaliaram-se as alterações no pH e cinética de fermentação ruminal de 9 bovinos, machos, com fístulas no rúmen. Os tratamentos foram: A = 0% CAI, B = 6,6% CAI e C = 15% CAI; a silagem de sorgo entrou na proporção de 70% em todos os tratamentos. As rações eram isonitrogenadas, com aproximadamente 12% de PB na MS. Houve diferença estatística (p 0,05) para a degradação de MS no tempo 48 horas (A = 54,4%; B = 54,2% e C = 58,7%), de PB às 12 horas (A = 40,3%; B = 47,7%; e C = 53,1%) e de FDA às 48 horas (A = 40,3%; B = 41,2% e C = 45,6%), ocorrendo maiores taxas de degradação com oaumento do nível de CAI na dieta. Os demais parâmetros (volume ruminal, turn over" do digesto ruminal e pH do conteúdo ruminal) não mostraram diferenças significativas entre tratamentos. O emprego crescente de CAI, até 15% da MS da ração, aumentou a degradação da MS, da PB e da FDA desse produto.