ABSTRACT
Orexins are newly discovered neuropeptides regulating feeding and vigilance and have been detected in neuroendocrine cells of the gut. Potential neuroendocrine functions of orexin are unknown. Therefore, the effects of orexin-A on the intestinal neuroendocrine cell line, STC-1, were investigated as a model system. RT-PCR demonstrated the presence of both OX(1) and OX(2) receptors. Stimulation with orexin-A produced a dose-dependent release of cholecystokinin (CCK), which was abolished by removal of extracellular Ca(2+) or the presence of the voltage-gated L-type Ca(2+)-channel blocker diltiazem (10 microM). Orexin-A (Ox-A) elevated intracellular Ca(2+), which was dependent on extracellular Ca(2+). Furthermore, orexin-A caused a membrane depolarization in the STC-1 cells. Ox-A neither elevated cAMP levels nor stimulated phosphoinositide turnover in these cells. These data demonstrate a functional orexin receptor in the STC-1 cell line. Ox-A produces CCK release in these cells, by a mechanism involving membrane depolarization and subsequently activation of L-type voltage-gated Ca(2+)-channels.
Subject(s)
Intracellular Signaling Peptides and Proteins , Receptors, Neuropeptide/biosynthesis , Receptors, Neuropeptide/chemistry , Animals , Calcium/metabolism , Carrier Proteins/metabolism , Cell Line , Cholecystokinin/metabolism , Cyclic AMP/metabolism , DNA Primers/chemistry , DNA, Complementary/metabolism , Diltiazem/pharmacology , Dose-Response Relationship, Drug , Inositol Phosphates/metabolism , Mice , Neuropeptides/metabolism , Orexin Receptors , Orexins , Patch-Clamp Techniques , Peptides/chemistry , Polymerase Chain Reaction , RNA, Messenger/metabolism , Receptors, G-Protein-Coupled , Reverse Transcriptase Polymerase Chain Reaction , Spectrometry, FluorescenceABSTRACT
In this study we have shown that Viable AC-2, a medium based on the ultrafiltrate fraction of bovine colostrum and adult bovine serum, can be used successfully as a fetal bovine serum (FBS) substitute in the culture of several anchorage-dependent and independent cell lines. Of the 15 cell lines cultured in 8% Viable AC-2 in microplates, 10 reached the maximum cell density of 65%-123% of that in 10% FBS, 4 cell lines reached maximum cell density of 35%-65% of that in 10% FBS and only one cell line, a human osteosarcoma G-292, grew slowly in Viable AC-2. In a small-scale suspension culture, 8%-15% Viable AC-2 supports the growth of Chinese hamster ovary cells (CHO-K1) on microcarriers in spinner flasks significantly better than 10% FBS. Shionogi mouse mammary tumour cell line (S115) transfected with human alpha 2-adrenergic receptor subtype C2 was used as a model to study recombinant protein production in Viable AC-2-supplemented medium. The results showed that in cell culture flasks and in an ACUSYST-R bioreactor, the alpha 2-C2 receptor expression level per mg of total protein was similar in both Viable AC-2 and FBS.