Increase in cytoplasmic cyclic AMP concentration stimulates Ca2' influx through the cyclic AMP-gated cation channel in the plasma membrane of cultured carrot cells. However, the Ca" current terminated after a few minutes even in the presence of high concentrations of cyclic AMP indicating that hydrolysis of the nucleotide is not responsible for stop of the Ca2' influx. Cyclic AMP evoked discharge of Ca*+ from inside-out sealed vesicles of carrot plasma membrane, and it was strongly inhibited when the suspension of the vesicles was supplemented with 1 PM of free Ca", while Cazf lower than 0.1 PM did not affect the Ca*'-release. The Ca '+ flux across plasma membrane was restored from this Ca2'-induced inhibition by the addition of calmodulin inhibitors or anti-calmodulin. These results suggest that Ca2' influx initiated by the increase in intracellular CAMP in cultured carrot cells is terminated when the cytosolic Ca" concentration reaches the excitatory level in the cells, and calmodulin located in the plasma membrane plays an important role in the response decay of the cyclic nucleotide-gated Ca" channel.

Thrombopoietin (TPO) is a cytokine which plays a central role in megakaryopoiesis and platelet production by binding to its cell surface receptor, termed c-Mpl. In the present study, two benzodiazepinones that compete with the binding of TPO to the extracellular region of c-Mpl were identified, and one of them stimulated the proliferation of a human TPOdependent megakaryocytic cell line, UT-7/TPO. It stimulated the activation of signal transducer and activator of transcription 5 in UT-7/TPO cells. These results suggest that a non-peptide compound can mimic the effect of TPO via c-Mpl.z 1998 Federation of European Biochemical Societies.

A random phage peptide library was constructed for the filamentous bacteriophage fuse5. The library was made by inserting a degenerate oligonucleotide which encodes 15 variable amino acids into the NH2-terminal region of the phage gene III protein. This library, containing 1x10(9) different phages, was screened with a human immunoglobulin fusion protein containing the extracellular region of human thrombopoietin receptor. Several phages were isolated following four cycles of enrichment and amplification. These phages specifically bound to the fusion protein. One phage peptide acted as an agonist of the thrombopoietin receptor, since it stimulated the proliferation of thrombopoietin-dependent cells and the differentiation of mouse bone marrow cells to megakaryocytes. The amino acid sequence of this peptide is not present in the primary amino acid sequence of thrombopoietin. This discovery may lead to the design of a small-molecular mimic of thrombopoietin.

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