A ring-deleted analog of atrial natriuretic factor--des[Gln18, Ser19, Gly20, Leu21, Gly22] ANF4-23-NH2 (C-ANF4-23)--binds with high affinity to approximately 99% of ANF receptors in the isolated perfused rat kidney. In this preparation, C-ANF4-23 is devoid of detectable renal effects and does not antagonize any of the known renal hemodynamic and natriuretic actions of biologically active ANF1-28. In contrast, both C-ANF4-23 and ANF1-28 increase sodium excretion and decrease blood pressure in intact anesthetized rats. This apparent contradiction is resolved by the finding that the ring-deleted analog markedly increases plasma levels of endogenous immunoreactive ANF in the rat. The results show that the majority of the renal receptors of ANF are biologically silent. This new class of receptors may serve as specific peripheral storage-clearance binding sites, acting as a hormonal buffer system to modulate plasma levels of ANF.

No abstract

Mice with a targeted deletion of ␤3 integrin were used to examine the process by which tumor cells metastasize and destroy bone. Injection of B16 melanoma cells into the left cardiac ventricle resulted in osteolytic bone metastasis in 74% of ␤3 ؉/؉ mice by 14 days. In contrast, only 4% of ␤3 ؊/؊ mice developed bone lesions. Direct intratibial inoculation of tumor resulted in marrow replacement by tumor in ␤3 ؊/؊ mice, but no associated trabecular bone resorption as seen in ␤3 ؉/؉ mice. Bone marrow transplantation studies showed that susceptibility to bone metastasis was conferred by a bone marrowderived cell. To dissect the roles of osteoclast and platelet ␤3 integrins in this model of bone metastasis, osteoclast-defective src ؊/؊ mice were used. Src-null mice were protected from tumor-associated bone destruction but were not protected from tumor cell metastasis to bone. In contrast, a highly specific platelet aggregation inhibitor of activated ␣IIb␤3 prevented B16 metastases. These data demonstrate a critical role for platelet ␣IIb␤3 in tumor entry into bone and suggest a mechanism by which antiplatelet therapy may be beneficial in preventing the metastasis of solid tumors. Bone metastases arise by means of a multistep process whereby tumor cells migrate from a primary tumor, disseminate through the arterial system to the bone marrow, and stimulate osteoclast (OC) activation and tumor-associated destruction of cortical and trabecular bone (1-3). Once tumor cells enter the bone marrow cavity, tumor-associated bone destruction (osteolysis) occurs in part through induction of host OC activation. Bisphosphonate OC inhibitors can partially decrease pain, fracture, and cord compression associated with bone metastases; however, Ϸ50% of bisphosphonate-treated patients can still develop new bone metastases and resultant skeletal complications (4-7). The role of the OC and OC inhibitors in preventing tumor entry into and attachment to bone is unclear.Venous injection models of ''metastasis'' have demonstrated that in mice platelet adhesion can play a role in tumor cell lung infiltration (8-15). Gasic et al. (8) first demonstrated that lowering the platelet count in mice resulted in decreases in lung invasion after i.v. injection of tumor cell lines. Antibodies directed against platelet antigens involved in tumor adhesion to platelets also decrease lung tumors in mice (9-13) after i.v. tumor cell injection. These observations have been made exclusively in model systems that examine tumor cell growth after passive tumor cell filtration by the pulmonary capillary bed. Furthermore, the molecular mechanisms underlying these phenomena have not been elucidated. Metastasis to other organs and bone could not be evaluated in these venous tumor cell injection systems. We present a report exploring the role of platelets in arterial-mediated metastasis to bone or other visceral organs.Cell adhesion receptors play roles at multiple stages of metastasis (16)(17)(18)(19). We sought to examine the role of host cell ␤ 3 -containing integ...

Up to 30% of acute myelogenous leukemia (AML) patients harbor an activating internal tandem duplication (ITD) within the juxtamembrane domain of the FLT3 receptor, suggesting that it may be a target for kinase inhibitor therapy. For this purpose we have developed CT53518, a potent antagonist that inhibits FLT3, platelet-derived growth factor receptor (PDGFR), and c-Kit (IC(50) approximately 200 nM), while other tyrosine or serine/threonine kinases were not significantly inhibited. In Ba/F3 cells expressing different FLT3-ITD mutants, CT53518 inhibited IL-3-independent cell growth and FLT3-ITD autophosphorylation with an IC(50) of 10-100 nM. In human FLT3-ITD-positive AML cell lines, CT53518 induced apoptosis and inhibited FLT3-ITD phosphorylation, cellular proliferation, and signaling through the MAP kinase and PI3 kinase pathways. Therapeutic efficacy of CT53518 was demonstrated both in a nude mouse model and in a murine bone marrow transplant model of FLT3-ITD-induced disease.

Recently, a second member of the protease-activated receptor (PAR) family, named PAR-2, has been identified. Similar to the thrombin receptor, PAR-2 appears to be activated by proteolytic-mediated exposure of a "tethered ligand" sequence and can also be activated by the corresponding synthetic peptides. Similarities in the amino acid sequence of the receptors' tethered ligand sequences suggest that their respective agonist peptides might not be absolutely specific for their particular receptors. To test this, the receptor specificity of each agonist has been determined by measuring the responses of Xenopus oocytes expressing the thrombin receptor or PAR-2 to agonist peptides or enzymes. Thrombin receptors responded to thrombin, the human thrombin receptor-activating peptide SFLLRNP-NH2 (TRAP) (EC50 = 0.1 microM), and Xenopus TRAP, TFRIFD-NH2 (EC50 = 1 microM), but did not show any increase in calcium efflux over control levels with trypsin (50 nM) or PAR-2 agonist peptides (100 microM). Human and murine PAR-2 receptors responded comparably to human and murine PAR-2 agonist peptides (SLIGKVD and SLIGRL, respectively) (EC50 = 0.5-2.0 microM) and trypsin, but not to thrombin. PAR-2 was also found to be responsive to TRAP (EC50 = 1 microM) but was unresponsive to Xenopus TRAP (50 microM). Responses to additional peptide agonist analogs suggest that an amino-terminal serine is critical for PAR-2 agonist activity.