Stimulation of voltage-dependent Ca2+channels by NO at rat myenteric ganglia

Im Rahmen dieser Arbeit wurde die Wirkung des nonadrenergen-noncholinergen Neurotransmitters Stickstoffmonooxid (NO) auf Zellen des Plexus myentericus der Ratte untersucht inklusive der Expression der Enzyme, die für die Produktion dieses Neurotransmitter verantwortlich sind. Myenterische Ganglien von 5 bis 12 Tage alten Ratten wurden enzymatisch isoliert und nach ca. 15 Stunden in Kultur mit dem Ca2+-Indikator Fura-2-AM beladen. Die intrazelluläre Ca2+-Konzentration ([Ca2+]i) wurde mit der Imaging Methode gemessen. Imaging Experimente zeigten, dass der NO Donor GEA 3162 (10-4 mol.l-1) einen signifikanten Anstieg der intrazellulären Calciumkonzentration verursachte. Dieser Anstieg ist durch Aktivierung spannungsabhängiger Ca2+-Kanäle verursacht, da der NO-Effekt in Abwesenheit von extrazellulärem Ca2+ oder nach Zugabe von Blockern von spannungsabhängigen Ca2+-Kanälen stark gehemmt war. Der Einfluss von GEA 3162 auf myenterische Zellen der Ratte scheint durch L- und N-Typ spannungsabhängige Ca2+-Kanäle vermittelt zu sein, denn die Hemmstoffe dieser Ca2+-Kanäle, Nifedipin bzw. w-Conotoxin, erwiesen sich als die wirksamsten, um den NO-bedingten Anstieg von ([Ca2+]i) zu vermindern. Polymerasekettenreaktions- (PCR-) und immunhistochemische Versuche wiesen die Existenz mehrerer Isoformen des Enzyms NO Synthase, nämlich der neuronalen Form (nNOS) und der endothelialen Form (eNOS), nach. Die dritte Isoform, die induzierbare NOS (iNOS), konnte nur mit immunhistochemischen Versuchen nach Stimulation der Ganglien mit dem proinflammatorischen Zytokin TNF-a nachgewiesen werden. Diese Versuche zeigen, dass NO unter physiologischen Bedingungen im Plexus myentericus gebildet werden kann.In this work I investigated the myenteric plexus of the rat small intestine with regard to the effects of the nonadrenergic-noncholinergic neurotransmitter, nitric oxide (NO). The action of NO on the intracellular calcium concentration [Ca2+]i of myenteric ganglionic cells was measured with imaging experiments; the presence of the enzyme responsible for synthesis of NO, i.e. the nitric oxide synthases, was investigated with polymerase chain reaction and immunohistochemical experiments. Myenteric ganglia from 5 - 12 days old rats were enzymatically isolated and kept for about 15 h under cell culture conditions. Changes in [Ca2+]i were measured by loading the ganglia with the Ca2+-indicator fluorescence dye, fura-2, and exposure to the NO donor, GEA 3162 (10-4 mol.l-1). The NO donor induced a pronounced increase in [Ca2+]i. This increase was reduced in the absence of extracellular Ca2+ and after exposure to blockers of voltage-operated Ca2+ channels, indicating that the effect of NO on [Ca2+]i is via activation of this type of Ca2+ channels. L- and N-type Ca2+ channels seem to play the dominant role by which NO induces changes in [Ca2+]i, since the blockers nifedipine and w-conotoxin caused the most effective inhibition in fura-ratio signal evoked by NO. PCR and immunohistochemical experiments revealed the presence of both neuronal and endothelial nitric oxide synthase isoforms in myenteric cells. The inducible isoform was shown by immunohistochemical experiments after incubation of the cells with the proinflammatory cytokine, TNF-a, indicating that NO is physiologically produced within myenteric ganglia

Biochemical and seminal parameters of lambs fed palm kernel cake under grazing system

ABSTRACT This study aimed to assess the effects of palm kernel cake on semen quality and biochemical parameters of Santa Inês lambs. A total of 40 animals with 24.10±2.72 kg body weight and five months old were assigned in a completely randomized design into four groups and 10 replicates. The animals were subjected to four levels of palm kernel cake (0, 15, 30, and 45%) based on dry matter. The trial lasted 90 days foregone by 15 days for adaptation. Blood samples were collected every 45 days from jugular vein using vacuum tubes without anticoagulant. Total serum cholesterol, triglycerides, high-density lipoprotein, low-density lipoprotein, and very-low-density lipoprotein were assessed. Once the animals reached puberty at a mean age of 225 days, the semen samples were collected by electroejaculator once a week for three sequence weeks and assessed for volume, color, aspect, wave motion, motility, sperm concentration, sperm vigor, total of spermatozoa per ejaculate, viable spermatozoa per mL, and sperm morphology. The data were subjected to analysis of variance and followed by regression analysis. Non-parametric data were analysed by Kruskal-Wallis test. Total cholesterol, high-density lipoprotein, triglycerides, and very-low-density lipoprotein were linearly increased. There was no difference for low-density lipoprotein. Diets did not affect mass motility, sperm motility, vigor, total spermatozoa per ejaculate, viability sperm per mL, and minor and total sperm defects. Sperm concentration increased linearly. Negative quadratic effects were observed for major sperm defects. Supplementation of diets with palm kernel cake up to 45% on dry matter enhance biochemical parameters and do not impair the qualitative variables of lamb sperm