The binding of glucagon to its hepatic receptor is known to result in a number of effects, including the intracellular accumulation of cAMP, the mobilization of intracellular Ca2+, and the endocytosis of glucagon and its receptor into intracellular vesicles. In this study, we begin to define the functional role of the COOH-terminal tail of the human glucagon receptor in glucagon-stimulated signal transduction and receptor internalization. We have created and expressed in Chinese hamster ovary (CHO) cells five truncation mutants in which the COOH-terminal 24, 56, 62, 67, and 73 amino acids have been removed. Cells expressing relevant truncated receptors were assayed for cell surface expression by immunofluorescence, for ligand-binding properties, for cAMP and Ca2+-mediated signal transduction properties, and for receptor endocytosis. In addition, a mutant receptor containing seven serine-to-alanine mutations in the COOH-terminal tail was studied. Our results reveal the following: 1) a region of the COOH-terminal tail that is required for proper cell surface expression, 2) the COOH-terminal 62 amino acids, which comprise the majority of the tail, are not required for ligand binding, cAMP accumulation, or Ca2+ mobilization, and 3) phosphorylation of the COOH-terminal tail is crucial for glucagon-stimulated receptor endocytosis.

Objective. To examine the regulation of intercellular adhesion molecule 1 (ICAM-1) in human synovial microvascular endothelial cells (HSE) and human umbilical vein endothelial cells (HUVE) upon exposure to a variety of agents.Methods. Cultured endothelial cells were treated with various cytokines alone and in combination. The expression of ICAM-1 was evaluated at several levels, including an investigation of messenger RNA (mRNA) and surface protein expression.Results. Treatment of HSE with interleukin-la (IL-la) or tumor necrosis factor a (TNFa) resulted in minimal increases in ICAM-1 expression, in contrast to findings with HUVE. Incubation of HUVE or HSE with IL-1 or TNF in combination with interferon-y (IFNy) greatly potentiated the increase in ICAM-1 surface expression. The synergistic effect of IFNy and TNF was confirmed by several methods, including a cell-based enzyme-linked immunosorbent assay, tluorescenceactivated cell sorting, immunofluorescence staining, and determination of mRNA levels. IFN y also augmented the actions of several other agonists on HSE, i.e., IL-4, lipopolysaccharide, and TNFPllymphotoxin. an identical manner. Unexpectedly, IFNy alone was a potent stimulus for HSE ICAM-1 mRNA synthesis, but was relatively ineffective in HUVE.Conclusion. These studies indicate that IFNy plays a critical synergistic role in the regulation of ICAM-1 expression in human synovial endothelial cells.

A flow cytometer was equipped with a modified sample station to facilitate online addition of mediators to the sample and reduce the time of delivery of the sample to the interrogation point. The ready availability of materials and straightforward nature of this design make modifications simple and facilitate measurements of cellular activation. Parameters such as pH, membrane fluidity, and calcium mobilization are easily measured in this system, because detection can be made less than 4 s after addition of mediator with no interruption of sample flow. The sample station modification is described in detail along with methods for mixing and temperature control.

A sample station modification previously described has been redesigned to provide greater flexibility and enhanced performance. The improved modification provides mixing and temperature regulation in a compact unit that mounts close to the nozzle holder for reduced transit times, allowing for addition of mediators to a sample in place on the flow cytometer, with observation of results in approximately 1 s. An electronic circuit activated at the time of injection generates full-scale pulses in the forward scatter channel. This provides the data with a time stamp for direct correlation of injection and cellular response. A detailed description of the modification, performance verification data, and practical applications in the measurements associated with cellular activation are presented.

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