Endothelin is a 21-amino acid peptide with remarkably diverse biological properties, including potent vasoconstriction, induction of mitogenesis, and a role in the development of blood vessels. In the present study, stimulation of the endothelin B receptor was found to activate three distinct mitogen-activated protein kinase signal transduction pathways, the extracellular regulated kinase (ERK) 2, c-Jun N-terminal kinase 1 (JNK), and p38 kinase. These mitogen-activated protein kinase isozymes are thought to mediate very different biological outcomes, suggesting that the observed pattern of kinases activation may be important for the diverse biological properties of endothelin. The cytoplasmic tail of the endothelin B receptor was found to be required for activation of all three mitogen-activated protein kinases and stimulation of intracellular calcium levels. An endothelin B receptor truncated at the C-terminal tail was not able to stimulate the mitogen-activated protein kinases or increase cytosolic free calcium. Furthermore, ectopic expression of the cytoplasmic tail attenuated signaling through the wild type receptor. The observed ERK activation appeared to be mediated by heterotrimeric G proteins, since ectopic expression of a transducin ␣-subunit inhibited endothelin-stimulated ERK activation. The data suggest that the cytosolic tail of the endothelin B receptor is involved in calcium mobilization and mitogen-activated protein kinase activation via a G protein-dependent mechanism. Endothelin (ET)1 is a 21-amino acid peptide with diverse physiological effects on cellular development, differentiation, vasoconstriction, and mitogenesis. There are three endothelin isoforms, ET-1, ET-2, and ET-3, that have preference for different endothelin receptor subtypes. The endothelin A (ETA) receptor binds ET-1, ET-2, and ET-3 with decreasing affinities, whereas the endothelin B (ETB) receptor binds ET-1, ET-2, and ET-3 with similar affinity; both are members of the G proteincoupled receptor (GPCR) superfamily (1-7).The putative ET receptor topology includes three extracellular domains, three intracellular cytosolic loops, and a cytoplasmic C-terminal tail, separated by seven highly hydrophobic regions thought to span the lipid bilayer. Ligand binding and specificity is conferred by the conformation of the extracellular portions of the receptor, whereas the intracellular domains physically interact with heterotrimeric G proteins to initiate signal transduction events. Receptor-G protein complexes are thought to form the high affinity ligand binding state of the receptor (8). For example, constitutively activated adrenergic and muscarinic receptor mutants have increased affinity for agonist with little effect on antagonist binding, supporting the view that the receptor-G protein complex is the high affinity binding complex (9, 10).Endothelin receptor mutagenesis studies suggest that residues within extracellular loop two are important for ligand binding. The intracellular domains of the receptor, thought to play a role in...

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A BSTRACT : Macrophages respond to infection or injury by changing from a "resting" cellular phenotype to an "activated" state defined by the expression of various cytotoxic effector functions. Regulation of the transition from a resting to an activated state is effected by cytokine and/or pathogenic signals. Some signals do not directly induce activation, but instead "prime" the macrophage to respond more vigorously to a second signal. One example of this priming phenomenon involves induction of nitric oxide (NO) synthesis by the enzyme nitric oxide synthase (NOS2). Our experiments indicate that low doses (1-5 Gy) of ionizing radiation can enhance the induction of enzymatically active NOS2 by IFN-␥ or LPS in J774.1 and RAW264.7 murine macrophage cell lines. Radiation alone did not produce this induction, rather, it was effective as a priming signal; cells exposed to radiation produced more NO when a second signal, either IFN-␥ or LPS, was applied 24 h later.

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