Abstract Thyrotropin-releasing hormone (TRH) reduces an inwardly rectifying K+ current in whole-cell voltage-clamped GH3 rat anterior pituitary cells. The TRH effect depends on the maintenance of a background level of Ca2+ in the pipette buffer, and is rapidly minimized by the intracellular dialysis produced under whole-cell conditions. Introduction of ADP-NH-P, a non-hydrolizable ATP analog, in the pipettes, nearly abolishes the TRH-evoked inhibition. The TRH-induced reduction of the inwardly rectifying current is significantly enhanced by incubation of cells 2–4 h with cholera toxin, but not by inclusion of 1 mM cyclic AMP in the pipettes. Under control whole-cell conditions, the reduction caused by TRH is not reversed upon washout of the neuropeptide. However, this effect is readily reversed by addition of purified catalytic subunits of protein phosphatase 2A (PP-2Ac) but not PP-1c to the buffer used to fill the patch pipettes. Among previous results with PP inhibitors, these data indicate that PP2A is involved in the phosphorylation/dephosphorylation mechanism(s) that regulate the delayed TRH effects on GH3 cell excitability.

Abstract Receptor tyrosine kinases (RTKs) are transactivated by the stimulation of G protein-coupled receptors (GPCRs). Sphingosine 1-phosphate (S1P), a ligand of GPCR, is known as a tumor-promoting lipid, but its signaling pathways are not fully understood. We here demonstrated that S1P induces rapid and transient tyrosine phosphorylation of epidermal growth factor receptor (EGFR) and c-Met in gastric cancer cells, both of which have been proposed as prognostic markers of gastric cancers. The pathway of S1P-induced c-Met transactivation is Gi-independent and matrix metalloproteinase-independent, which differs from that of EGFR transactivation. Our results indicate that S1P acts upstream of various RTKs and thus may act as a potent stimulator of gastric cancer.

Abstract Four proteins of the lipocortin family, lipocortin I (35 kDa), lipocortin II (36 kDa), lipocortin V (32 kDa) and lipocortin VI (67–70 kDa), were identified in the cytosols of 2-day-old cultures of thyroid cells. Only lipocortin I was phosphorylated in vitro in fully differentiated, thyroid stimulating hormone-treated cells (0.1 mUml). Protein kinase C was the only kinase activity which phosphorylated lipocortin I. Phosphorylation shifted its pI from 6.9 to 6.6. The in vitro phosphorylation of lipocortin I was impaired in cultures exposed for 2 days to phorbol ester (10−7 M), although it was present in both the cytosol and the particulate fraction of these cells.

Abstract The effect of tumor necrosis factor on the oxidative modification of LDL by U937 human monocytes or murine endothelial cells was studied by determination of the lipid peroxidation product content and the electrophoretic mobility of the particle. In the range of concentrations from 2.5 to 10 ngml, the cytokine induced a dose-dependent increase in cellular-induced oxidation of LDL. This effect was accompanied by a stimulation of LDL degradation by J774 macrophage-like cells. Concurrently, the TNF-treated cells secreted superoxide anion with a higher rate. Since LDL oxidation is believed to be an inportant feature in the formation of the atherosclerotic plaque, the described effects of TNF might be of importance in long-term exposure to this cytokine during inflammation.

Abstract Stably transfected PC12D cell lines overexpressing a catecholamine biosynthesis regulatory protein, V-1, were used to examine the functional role of V-1 in catecholamine secretion. High K+-induced dopamine secretion in V-1 overexpressing clones was shown to be markedly potentiated compared with control clones carried with a vector alone. As assayed intracellular calcium concentration ([Ca2+]i) using fura-PE3, V-1 overexpression was observed to enhance high K+-elicited [Ca2+]i elevation. Electron microscopic analysis revealed an increase in dense-cored vesicle formation by V-1 overexpression. These results suggest that the enhancement of high K+-induced dopamine secretion by V-1 overexpression results from the potentiation of high K+-induced [Ca2+]i elevation and the increase in the number of dense-cored vesicles.

Abstract Cell crawling entails the co-ordinated creation and turnover of substrate contact sites that interface with the actin cytoskeleton. The initiation and maturation of contact sites involves signalling via the Rho family of small G proteins, whereas their turnover is under the additional influence of the microtubule cytoskeleton. By exerting relaxing effects on substrate contact assemblies in a site- and dose-specific manner, microtubules can promote both protrusion at the front and retraction at the rear, and thereby control cell polarity.

Abstract The myo-inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ mobilization in single saponin-permeabilized and fura-2-loaded parietal cells was analysed by a fluorescence digital image processor. When the cells were incubated with ATP, free cytoplasmic Ca2+ concentration ([Ca2+]i) increased in some restricted cytoplasmic regions showing discontinuous plateau and in the peripheral cytoplasm showing continuous [Ca2+]i gradient towards the plasma membranes. When treated with IP3, the high plateau enlarged to the entire cytoplasm and (a) new higher plateau(s) appeared and enlarged again in a transient manner. The IP3-induced Ca2+ transient was also observed by fluorescence microphotometry of the single cells or by fluorescence spectrophotometry and 45Ca2+ uptake experiment of the cell population.

Abstract Five-lipoxygenase-activating protein (FLAP) is usually described as an essential protein to activate the leukotriene (LTs) synthesis via the 5-lipoxygenase pathway. In the enterocyte model HT29 cl.19 A cell line, 5-lipoxygenase metabolism was found despite the lack of FLAP expression. Therefore HT29 cl.19A represents an original mammalian model to study FLAP-dependent leukotriene synthesis. In FLAP cDNA transfected HT29 cl.19 A cells, FLAP expression led to an increase in cyclooxygenase pathway products (mainly PGE2) without an increase in 5-lipoxygenase metabolism. This increase in PGE2 synthesis was associated with a cyclooxygenase-2 up-regulation in comparison to untransfected HT29 cl.19A cells. These results suggest a possible interaction between the two major pathways of arachidonic acid metabolism.

Abstract The Ca2+ receptor mechanism of the parathyroid cell was studied using La3+ as a probe. La3+ was found to bind to the cell surface without further penetration. Measurements of 45Ca fluxes and the cytoplasmic Ca2+ concentration (Cai2+) revealed a stimulatory component in the action of La3+ on Ca2+ permeability resulting in a rise in Cai2+ These effects mimicked those obtained when raising the extracellular Ca2+ concentration from 0.5 to 3.0 mM, but the actions of La3+ and Ca2+ were not additive. The results suggest the existence of a novel Ca2+ permeability physiologically activated by binding of Ca2+ to an external receptor.

Abstract We studied the effects of cytostatic drugs on porcine coronary artery spindle-shaped (S) and rhomboid (R) smooth muscle cell (SMC) biological activities related to intimal thickening (IT) formation. Imatinib, and to a lesser extent curcumin, decreased proliferation of S- and R-SMCs and migratory and urokinase activities of R-SMCs more efficiently compared with cyclosporine plus rapamycin. Imatinib increased the expression of α-smooth muscle actin in both SMC populations and that of smoothelin in S-SMCs. It decreased S100A4 expression in R-SMCs. By promoting SMC quiescence and differentiation imatinib and curcumin may represent valid candidates for restenosis preventive and therapeutic strategies.