A growth factor stimulating DNAsynthesis of adult rat hepatocytes in primary culture was found in the conditioned medium after culturing nonparenchymal liver cells (NPC). Adding heparin to the NPC cultured medium stimulated the growth-factor secretion from NPC.The growth factor was secreted mainly by Kupffer cells. The partially purified growth factor from the NPC appeared to be related to the HGF isolated from platelets according to three criteria: (a) binding to Heparin-Sepharose and eluting at about 0.65 M NaCl, (b) having a Mr of about 70 kDa, (c) having an immunoreactivity to antibody against rat platelet-derived HGF. Adding heparin to the NPC cultured medium also resulted in protection of the growth factor from heat- and acid-inactivation, but the direct interaction of heparin with the partially purified growth factor did not lead to such protection. Per fusion of normal adult rat livers with Hanks' solution containing 1 M NaCl in situ led to the release of large amounts of hepatocyte growth factor (10). These findings suggest that the hepatocyte growth factor derived from NPC binds to ECM between the layer of hepatocytes and endothelial cells forming the sinusoids in normal adult rat liver and that this may play a role in stabilization and maintaining the pool of HGF, which functions to constantly supply HGF in the setting of liver regeneration.
We studied whether there is a quantitative relationship between free cytosolic Ca++ levels and the release of an endothelium-derived relaxing factor (EDRF) from cultured fetal bovine aortic endothelial cells (EC). EC pretreated with indomethacin were stimulated by the agonists adenosine triphosphate (ATP), bradykinin (BKN), acetylcholine (ACh) and calcium ionophore (A23187) in various concentrations (10-8-10-5 M), and the amount of EDRFreleased was determined on the basis of endothelium-free rabbit aortic ring relaxation and cultured smooth muscle cell CGMPcontent. Changes in intracellular Ca++ concentration ([Ca++]i in response to the same stimuli were determined by photometric fluorescence microscopy using the fluorescent calcium indicator Fura-2. EC stimulation by ATP and A23187 induced dose-dependent increases in both [Ca++]i and the amount of EDRFreleased. BKNincreased both [Ca++]i and EDRFrelease upon initial exposure (10-8M), but there were no further changes at higher concentrations. AChinduced no significant changes in either [Ca++]i or EDRFrelease. There was a close quantitative correlation between agonist-induced changes in [Ca++]i and the amount of EDRFreleased (relaxation response in aortic rings and CGMPlevels.) (p < 0.001) Removal of extracellular Ca++ eliminated continuous elevation in both [Ca++]i and the amount of EDRFinduced by ATP (10-5 M), BKN(10-8M) and A23187 (10-6M). These findings suggest that intracellular Ca++ levels are directly linked to the amount of EDRFreleased, and that extracellular Ca++ is essential for EDRFrelease because its influx is involved in the continuous elevation of [Ca++]i.
Epidermal growth factor and transforming growth factor alpha stimulated DNA synthesis in primary cultures of adult rat hepatocytes. In the presence of neurotensin, the stimulatory effect of epidermal growth factor or transforming growth factor α on DNA synthesis was enhanced 3- to 5-fold. Since neurotensin by itself did not stimulate DNA synthesis at all concentrations tested, enhancement of DNA synthesis by neurotensin is due to the amplification of the effect of epidermal growth factor or transforming growth factor a. The amplification by neurotensin was observed in a dose-dependent manner with a maximal effect at 10-8 M, although its effect was significantly observed at as low as 10-10 M. This neurotensin amplification of DNA synthesis was observed when hepatocytes were cultured in Williams' medium E, but not in Leibovitz L-15 medium, suggesting that minor components in the mediumare required for hepatocytes to fully respond to neurotensin. Neurotensin-related peptides such as kinetensin or neuromedin-N did not have this amplifying effect on DNA synthesis at any concentration tested. From these results, neurotensin can be regarded as a new secondary mitogen.
The development of a spatio-temporal pattern of Ca2+ concentration (Ca2+ pattern) in the plasmodium of Physarum polycephalum during repulsive response was studied using fura-2. In the migrating cell, the gradient of the Ca2+ concentration (Ca2+ gradient) immediately showed a decrease in local concentration in the area (S-site) stimulated by 50 mM KCl. The concentration rose and then decreased in a site neighboring the S-site. This transient increase of Ca2+ concentration, the duration of which was approx. 10 minutes, was propagated to the site most distant from the S-site. There, the Ca2+ concentration gradually rose and remained at a high level. Twenty-five minutes after stimulation, a new Ca2+ gradient was established throughout the plasmodium. The migratory direction of the cell as a whole then changed. In this process, although the period of Ca2+ oscillation changed at the S-site, this change was only local to the site. During the information processing of the local repulsive stimulus, the transient Ca2+ increase propagated the local information about the stimulus to the non-stimulation sites (NS-sites), leading to the generation of a new pattern and the start of coordinated migration of the plasmodium.
Hepatocyte growth factor (HGF) is known to induce the dispersion of epithelial cells, as scatter factor. On the other hand, cadherins play a crucial role in connecting cells together. Twogroups of cadherins are involved in epithelial cell adhesion, those locating in adherens junctions (AJ) and in desmosomes. Here, we examined the effect of HGF on the function of these cadherins in keratinocyte cell lines F and 308R, which expressed E- and P-cadherin in AJ (referred to as AJ cadherins) and desmoplakin in desmosomes. In the presence of HGF, these cells spread more extensively than in control cultures and their associations apparently loosened. However, they maintained cell-cell contacts where cadherins and desmoplakin concentrated, although the level of the concentration was reduced by HGF treatment. When antibodies to E- and P-cadherins were added to cultures of these cells without HGF, AJ cadherins were redistributed into non-junctional areas of the cells, but desmoplakin still localized at cell-cell boundaries. When HGF was added together with anti-AJ cadherin antibodies to the cultures, cell-cell contacts were now disrupted. In these cultures, not only AJ cadherins but also desmoplakin were lost at cell-cell contact sites, indicating that HGF can disrupt desmosomal cell-cell adhesion when AJ cadherins are inactive. These results suggest that, although HGF cannot block cadherin-mediated cellcell adhesion when the entire cadherin system is intact, it might modulate the activities of cadherins, especially, of desmosomal cadherins.
In this study, we measured hydrogen peroxide (H2O2) release as one of the functions of mature eosinophils, and utilized it as a quantitative index. Wedemonstrated that 1) the human eosinophilic leukemia cell line, EoL-1, did not release H2O2 when stimulated with phorbol myristate acetate (PMA), but after culturing with tumor necrosis factor (TNF) and interferon-γ (IFN-γ) it acquired the ability to release H2O2; 2) the ability to release H2O2 was time dependent and reached a peak after 4 days of culture; 3) administration of TGF-β or GM-CSF, with TNF and IFN-γ enhanced the PMA-induced release of H2O2 from EoL-1. To examine the potential relationship between c-myc gene expression and induction of the ability to release H2O2, Northern analysis of c-myc gene expression in EoL-1 cocultured with TNF and IFN-γ was performed. The results showed that the c-myc gene was spontaneously expressed in EoL-1, and the level of c-myc mRNA was markedly reduced after the cells were cocultured with TNFand IFN-γ, suggesting that the decrease of the c-myc mRNA level is closely associated with induction of the ability to release .